In multiple sclerosis, impaired oligodendrocyte differentiation limits remyelination and leads to axonal atrophy and neurodegeneration. Identified in several compound screens, non-selective muscarinic receptor (MR) antagonists improve myelin repair in rodents. Understanding the mechanisms by which these antagonists act and the role of muscarinic acetylcholine (ACh) signaling in OPCs is of paramount importance to the clinical translation of this approach. Importantly, our preliminary studies provide direct evidence for a role of M3R function in OPC differentiation. Using a novel combination of lentiviral M3R KD and human OPC transplantation, we found that ex vivo M3R KD increases oligodendrocyte production following engraftment in shiverer/rag2 mice, a preclinical model of childhood hypomyelination. Furthermore, we show that conditional M3R knockout increased OPC differentiation following spinal cord demyelination. While these data establish a clear functional role of M3R in OPCs, that cannot be compensated by other MR subtypes, there are gaps in knowledge regarding the potential of M3R targeting to improve functional and structural myelin repair, the role of MR-induced store-operated calcium-entry (SOCE), and the cellular sources of ACh following demyelination. The three aims in this proposal address related aspects of MR signaling to better define and characterize therapeutic targets to improve myelin repair. Aim 1: Determine the importance of M3R signaling in human OPCs following transplantation and in resident OPCs during remyelination in adult CNS. We will test the hypothesis that blocking M3R signaling in OPCs will augment and improve pre-clinical outcomes of transplant-mediated repair and enhance spontaneous remyelination. Aim 2: Determine the mechanisms by which M3R regulates differentiation of hOPCs. We will test the hypothesis that MR signaling is mediated by oscillatory SOCE. We will use optogenetic SOCE (OptoSTIM1) and conditional deletion of Stim1 to determine the functional role of SOCE in OPCs as an effector of M3R signaling and during remyelination. Aim 3: Determine the functional sources of ACh during remyelination. We will test the hypothesis that ACh synthesized by infiltrating T cells and neighboring cholinergic fibers impair OPC differentiation and remyelination. We will use ChAT-GFP reporter and ChAT floxed mice to characterize and then ablate ChAT expression from demyelinating spinal cord lesions. In summary, this project will establish the therapeutic utility of specifically targeting M3R to improve myelin repair, the mechanisms by which M3R acts to block OPC differentiation, and the functional sources of ACh during remyelination. In addition to the now-established role of activity-dependent differentiation and myelination, these studies will begin to characterize the novel concept that some neurotransmitters such as ACh act to prevent untimely OPC differentiation. Lastly, by establishing that M1/3R acts primarily via SOCE, we will provide proof-of-concept that SOCE could itself be a common mechanism by which OPC differentiation is pathologically delayed.